This article was submitted to Biosafety and Biosecurity, a section of the journal Frontiers in Bioengineering and Biotechnology
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High containment biological laboratories (HCBL) are required for work on Risk Group 3 and 4 agents across the spectrum of basic, applied, and translational research. These laboratories include biosafety level (BSL)-3, BSL-4, animal BSL (ABSL)-3, BSL-3-Ag (agriculture livestock), and ABSL-4 laboratories. While SARS-CoV-2 is classified as a Risk Group 3 biological agent, routine diagnostic can be handled at BSL-2. Scenarios involving virus culture, potential exposure to aerosols, divergent high transmissible variants, and zoonosis from laboratory animals require higher BSL-3 measures. Establishing HCBLs especially those at BSL-4 is costly and needs continual investments of resources and funding to sustain labor, equipment, infrastructure, certifications, and operational needs. There are now over 50 BSL-4 laboratories and numerous BSL-3 laboratories worldwide. Besides technical and funding challenges, there are biosecurity and dual-use risks, and local community issues to contend with in order to sustain operations. Here, we describe case histories for distinct HCBLs: representative national centers for diagnostic and reference, nonprofit organizations. Case histories describe capabilities and assess activities during COVID-19 and include capacities, gaps, successes, and summary of lessons learned for future practice.
“Chance favors the prepared mind, and opportunity favors the bold.”― Louis Pasteur
The field of global health security, while diverse in its methodology and applications, can be simply delineated by the prevent-detect-respond model. Major activities are performing research and development (R&D), especially those involving international cooperation, and conducting infectious disease surveillance; both enhance global health security and specifically biosecurity by upholding the Biological Weapons Convention (BWC) and the United Nations Security Council Resolution (UNSCR) 1,540 that prohibit the use of biological weapons and are legally binding for all countries. R&D and infectious disease surveillance activities also support globally recognized frameworks such as the International Health Regulations (IHR) 2005 and the Global Health Security Agenda (GHSA), which both strengthen human and veterinary health systems. As a whole, a web of prevention model illustrates a framework of these major instruments categorized under biosafety and biosecurity (
At its core, biological containment refers to primary containment such as equipment such as biosafety cabinets (BSC) used in HCBLs to prevent contamination of the sample of interest and protect the people handling those materials and secondary containment related to infrastructure (2, 3. High containment biological laboratories (HCBL) refer to the biosafety level-3 (BSL-3) and -4 (BSL-4) laboratories which are designed to contain pathogens to prevent their release into the environment and provide a safe setting to protect those working with these pathogens (
Risk-based assessments continue to be objective approaches to ensure biosafety and biosecurity in HCBLs when performing tasks with agents and material such as new and emerging viruses that are not fully characterized (
HCBLs play a critical role in rapid advancement of research to characterize human and animal pathogens, assist in disease surveillance, and conduct the initial pre-clinical research that sustains the pipeline for development of diagnostics, therapeutics and vaccines. Moreover, the research conducted in HCBLs enables improved understanding of the pathogen and in turn informs the necessary public health measures to control and prevent further transmission and disease pathogenesis. The scientific capabilities of the highly trained personnel working in HCBLs are a tremendous resource not only because of the research they conduct, but also because they can provide critical guidance on how to effectively manage a highly infectious pathogen at lower containment levels if necessary. The COVID-19 pandemic has demonstrated the importance of HCBLs, their associated resources, and trained personnel in combatting epidemics caused by emerging pathogens.
HCBLs have evolved in terms of infrastructure, physical controls, policies, human resources, and workforce. Biodefense in the context of countering biowarfare and bioterrorism developed and grew as a market and enterprise following September 11, 2001 and the anthrax letter attacks. In addition, interest in HCBLs has resulted in growth in number of BSL-3 and BSL-4 laboratories worldwide and subsequent oversight (
Several motivations for building HCBLs exist including diagnostic needs, enhancing infectious disease surveillance capability and sustainability, promoting growth for life science and biotechnology, and improvements in scientific research capabilities (
Although CDC and WHO recommend that clinical samples such as confirmed and suspected COVID-19 specimens can be handled in BSL-2 facilities for routine diagnostic purpose, isolation or propagation of high concentrations of viruses should be conducted in BSL-3 at minimum (
While the HCBLs described may have different missions, all of them adhere to international norms and instruments discussed earlier as well as national and local regulations. Two HBCLs have bilateral partnerships with the US Defense Threat Reduction Agency’s Biological Threat Reduction Program (BTRP) which include agreements that adhere to the BWC and UNSCR 1540. The partnerships focus on biosafety and biosecurity, infectious disease surveillance and cooperative research. Our case histories describe capabilities and assess activities during COVID-19 and include capacities, gaps, successes, and summarize lessons learned. For example, one state public health laboratory in the US successfully increased laboratory capacity by cross-tasking personnel already trained in molecular extraction and who were already respirator fit-tested to work in BSL-3 (
Summary of high-containment biological laboratories (HCBL) described in case histories. HCBLs from three continents are listed.
HCBL | Organization type | Description | |
---|---|---|---|
BSL-3 | Kazakhstan Central Reference Laboratory | National laboratory | Crucial functions ( |
• Repository for especially dangerous pathogen | |||
• Reference, research, and training center | |||
• Promotes international laboratory practices | |||
MRIGlobal | Non-profit research | Accreditations and registrations ( |
|
• CDC registered for Select Agent and Toxins | |||
• CAP, CLIA certified laboratories | |||
• ISO-9001:2015, ISO 17025:2005 | |||
Taiwan Centers for Disease Control (CDC) | National Laboratory | Relevant mission ( |
|
• National reference and research center | |||
• Promotes international laboratory practices | |||
BSL-4 | International Center for Medical Research of Franceville (CIRMF) | National laboratory | Crucial roles |
• National diagnostic center and reference laboratory | |||
• Regional center for diagnosis of pathogens including bacteria (anthrax) and viruses: CCHF, rabies, SARS-CoV-2, Ebola, Marburg, polio ( |
|||
• Recognized international center for research |
As part of the legacy United States (US) Cooperative Threat Reduction program and long-standing partnership with Kazakhstan, DTRA BTRP funded and commissioned the Central Reference Laboratory (CRL) located in Almaty on the National Scientific Center for Especially Dangerous Infections named after M. Aikimbayev (NSCEDI). The CRL validation was completed in August 2017, and subsequently on September 29, 2017, the facility was transferred to the Government of Kazakhstan. Several cooperative research projects were also funded to support studies that the CRL would eventually house and perform (
With its capacity, capability, and national stature, the CRL serves as the leading diagnostic laboratory in Kazakhstan. It maintains the national collection of strains of especially dangerous pathogens or EDP from the Ministry of Health (MoH) and Ministry of Agriculture (MoA) and has organizational and methodological functions including informatics and analysis typical of similar public health laboratories. The CRL facility operates under NSCEDI (MoH) management, with MoA and Ministry of Education and Science (MoES) assigned rent-free space in the facility, with dedicated BSL-2 laboratories. The MoA also shares BSL-3 and ABSL-3 laboratory space with the MoH. By agreement, MoH, MoA, and MoES are collaborating through consolidated efforts and resources. So far, over 100 CRL specialists have been trained, half of which are NSCEDI employees, 25 are MoES employees, and 25 are with MoA staff. The CRL is becoming a regional center of international cooperation for joint research, and professional training in with partners including Armenia, Azerbaijan, Georgia, Kyrgyzstan, Mongolia, Russia, Tajikistan, Turkmenistan, and Uzbekistan.
The CRL also implemented a quality management system in accordance with the requirements of ISO 9001:2015 Quality Management Systems. DEKRA (
During the COVID-19 pandemic, DTRA funded laboratories including the CRL became active for diagnostic testing and related R&D. In May 2020, the International Center for Vaccinology at the Kazakh National Agrarian Research University (KazNARU) and the NSCEDI developed a national vaccine against SARS-CoV-2 virus infection (
On August 28, 2020, KazNARU and The Ohio State University, USA also generated a vaccine based on chitosan with mannose nanoparticles for intranasal application (NARUVAX-C19 Nano). Recombinant SARS-CoV-2 Spike/RBD protein was used as an antigen. Testing of this vaccine in laboratory animals is started in mid-September 2020 in the CRL’s ABSL-3 laboratory. In early 2021, research for a hamster model to evaluate the effectiveness of SARS-CoV-2 vaccine candidates was completed. The SARS-CoV-2 hamster challenge model confirmed immunogenicity of our vaccines and verified their protective efficacy against intranasal infection with D614G spike variant of SARS-CoV-2.
On August 14, 2020, KazNARU, NSCEDI, and TreeGene LLP, used a GISAID platform (
In November 2020, the isolated and characterized 3 strains (hCoV-19/Kazakhstan/KazNAU-NSCEDI-5/2020, Accession ID: EPI_ISL_514126; hCoV-19/Kazakhstan/KazNAU-NSCEDI-7/2020, Accession ID: EPI_ISL_514127; hCoV-19/Kazakhstan/KazNAU-NSCEDI-481/2020, Accession ID: EPI_ISL_514093) of SARS-CoV-2 were deposited in the Republican Collection of Microorganisms and Depository of Especially Dangerous Infections of the NSCEDI. These strains are used to test diagnostic test systems and vaccines and to determine the antiviral activity of various substances
On March 19, 2021 the Minister of Health of Kazakhstan reported COVID-19 positive samples from Almaty that were found to have mutations of representing the Alpha and Beta variants of SARS-CoV-2. The International Center for Vaccinology of KazNARU and the Scientific and Practical Center for Sanitary and Epidemiological Expertise and Monitoring of the Ministry of Health of the Republic of Kazakhstan confirmed the presence of mutations in these samples. KazNARU, Kazakhstan-Japan Innovation Center, and TreeGene performed sequencing of the complete S gene. The results showed that in one sample, the S gene contained amino acid substitutions 501Y, 570D, 614G, 681H, 716I, 982A, 1118H, and amino acid deletions H69, V70, Y144, which are all typical of the Alpha variant B.1.1.7 (posted at GISAID on March 25, 2021). In the second sample, the S gene contains amino acid substitutions 80A, 215G, 242H, 417N, 484K, 501Y, 614G, 701V, 1078S, amino acid deletions A243, L244, H245, which are all typical of South African variant B.1.351 (posted on GISAID March 25, 2021). The NSCEDI and KazNARU research team isolated these virus variants in their BSL-3. Availability of these variants will allow Kazakhstani scientists to evaluate the effectiveness of both domestic and foreign vaccines and drugs against COVID-19.
For-profit companies, non-profit organizations, as well as national laboratories often employ personnel with expertise in diverse fields such as chemistry, engineering and biology who perform contract research and development, test and evaluation and related work. These organizations also invest heavily in their facilities and equipment as part of growing their core competencies to support their respective client’s project work. MRIGlobal is a non-profit research organization that provides scientific solutions in a range of disciplines to commercial and government clients. Investment in staffing resources and quality management systems provided some of the foundational core competencies that lead to MRIGlobal’s work with pathogens, obtaining a Biological Select Agent and Toxins (BSAT) registration through the US CDC’s Federal Select Agent Program, and related biosafety and biosecurity standards. All BSAT work performed at MRIGlobal is done in compliance with select agent code of federal regulations (CFR) which are 7 CFR Part 331, 9 CFR Part 121 and 42 CFR Part 73.
MRIGlobal’s BSAT program maintains compliance with the regulations described in their corporate Biological Safety Plan, Biosecurity Plan, and Incidence Response Plan (IRP). MRIGlobal also maintains numerous operational SOPs related to the handling, storage and disposal of BSATs which are regularly reviewed and updated according to guidance on BSAT. There is also a personnel reliability program employed for all staff that have access to and work with BSAT.
Prior to initiating any laboratory work with infectious biological materials, MRIGlobal requires a review and approval of the planned project work by the Biosafety Officer (BSO) and the Biosafety Review Committee (BRC). The BRC reviews all new projects involving biological materials, toxins, recombinant deoxyribonucleic acid (rDNA), or synthetic nucleic acid molecules, to determine any potential safety issues. The review includes an assessment of the agents that will be worked with, the laboratory space that will be used for the work, the relevant personnel, and their laboratory experience. In addition, a Hazard Analysis is performed to identify any potential safety issues and ways to mitigate the risk to ensure the safety of laboratory personnel.
Initially during the COVID-19 pandemic, one of MRIGlobal’s BSL-3 facilities was designated as the laboratory space dedicated to working with the SARS-CoV-2 virus. As the demand for work increased, so did the utilization of the BSL-3, which decreased the availability of Class II BSCs. For example, existing non-COVID-19 project work with BSAT and Risk Group 3 agents was being performed in parallel. According to a risk assessment for handling high and low concentrations of virus, MRIGlobal converted a BSL-2 microbiology laboratory space into a BSL-2 enhanced, or BSL-2 plus (BSL-2+), laboratory space for activities using low concentrations of virus. This is common practice in which the BSL-2+ laboratory is essentially a BSL-2 laboratory with some BSL-3 features and additional safety equipment, in this case a requirement for respiratory protection and an area for donning and doffing of personal protective equipment (PPE) were established (
Any work with SARS-CoV-2 virus or nucleic acids is performed in a BSC in the BSL-2+ laboratory to prevent exposure to the virus through spills or aerosolization. Waste materials from the BSL-2+ are designated as medical waste and are removed from the laboratory space for incineration off-site. Typically, SARS-CoV-2 materials are bleach inactivated prior to removal from the laboratory space. The implementation of these enhanced procedures and staff training resulted in the successful conversion of a BSL-2 laboratory into BSL-2+ laboratory space. This resulted in doubling the number of BSCs available for handling and processing SARS-CoV-2. MRIGlobal was able to designate this converted space for project work with the virus at near-limit of detection (LOD) levels, while work with high-titer virus stocks was reserved for the BSL-3 facility. A proper risk assessment demonstrated the BSL-2+ can serve as an effective model for laboratories looking to increase available laboratory space for use with non-BSAT infectious pathogens. Current guidance from the CDC limits these efforts at BSL2+ with newly emerging SARS-CoV-2 variants and requires work with variants to be performed at BSL-3 (
The primary mission of the CIRMF BSL-4 laboratory is to diagnose suspected cases of hemorrhagic fever and investigate related cases in the Central Africa region, which has experienced outbreaks of Ebola and other hemorrhagic fever pathogens. Ebolavirus disease is a hemorrhagic disease caused by one of the most feared and virulent pathogens currently threatening human population (
Following the Mayibout outbreak, the Emerging Viral Disease Unit (UMVE) was set up in 1998 to study the emerging Ebolavirus disease. The CIRMF BSL-4-like, which UMVE managed and operated, is now managed by the Department of Virology. Many researchers, technicians and engineers as well as agents of the Ministry of Health of Gabon have been trained and empowered to work in this laboratory. Today four people, all working at CIRMF, are the only ones authorized to enter and to work there. They are also in charge of training collaborators whose research requires protocols to be developed in high containment environments. Since the beginning of its construction, almost all suspected or sporadic cases of the human and animal epidemics from 1997 to 2019 in the Central African sub-region have been diagnosed in this laboratory. The glove box and equipment such as climatic chambers, heat chambers and incubators allowed for the isolation and the culture of many variants. Thus CIRMF maintains a stock of samples from past epidemics of Ebola virus and Marburg virus strains isolated from both humans and great apes, and strains of rhabdoviruses, Crimean Congo Hemorrhagic Fever virus and poliovirus. Nevertheless, all poliovirus strains were destroyed according to the WHO procedures in the Polio eradication program.
Because of its ability to respond against hemorrhagic fever epidemics especially Ebolavirus disease, that occurred in various countries of the Central African Region, the CIRMF BSL-4 was designated as a WHO Collaborative Centre for the diagnosis of hemorrhagic fevers in 2016–2020. Thus, the laboratory fully adhered to WHO guidelines. Today, CIRMF continues a partnership with the DTRA BTRP where Sandia National Laboratory performed a risk assessment in order to update all the guidelines regarding the biosafety and the biosafety. Further assessments are planned at CIRMF to attain ISO:35001.
On January 31, 2020, the WHO declared COVID-19 as a global health emergency after SARS-CoV-2 spread in almost all continents. Fearing a rapid spreading throughout the African continent, the Africa Centres for Disease Control and Prevention (Africa CDC) chose the CIRMF among the reference laboratories for the diagnosis of suspected COVID-19 cases. CIRMF had the infrastructure including BSL-3 and BSL-4 laboratories with the scientific expertise, reputation, and experience managing Ebola diseases and arboviruses outbreaks (
As part of their mission, the Taiwan Centers for Disease Control (Taiwan CDC) has engaged with international partners to perform activities aligned with WHO and IHR for performing Joint External Evaluations of their health systems and also developed national legislature for regulating high containment laboratories, biosafety, and managing select agents and toxins (
The performance of these designated laboratories was fully demonstrated during the global outbreaks of influenza A virus subtype H7N9, MERS-CoV, and Zika viruses in 2014, 2015, and 2016, respectively. The authorized laboratories are also responsible for the diagnosis of categories 2, 3, and 4 diseases in BSL-2 or BSL-2 laboratories outfitted with negative air pressure facilities (
The commissioned laboratories are responsible for surveillance of important communicable diseases in the community. Clinical isolates of pathogens such as influenza, enteroviruses, and tuberculosis are routinely collected by the commissioned labs and sent to Taiwan CDC for detailed genetic, antigenic, and resistance analyses. All of the laboratories in the network are qualified through annual proficiency testing, and Taiwan CDC consistently monitors their diagnostic qualifications. Taiwan CDC and local health bureaus also provide periodic training in biosafety and technical hands-on procedures. For BSL-3 laboratory workers, the minimal requirement of biosafety and biosecurity training for each new employee of microbiological laboratories is 15 hours. After the first year, employees should also receive at least a 4-h refresher training course in each of the following years. The government of Taiwan pays the diagnostic fees in full. For example, the diagnosis of some category five diseases, such as those caused by influenza A (H7N9) and SArS-CoV-2 costs 3,000 New Taiwan dollars (NT$) per test which is approximately $100 US dollars.
From this experience, Taiwan recognizes that diagnostic testing is key to containing COVID-19. At the beginning of the COVID-19 outbreak, eight medical centers with experience in emerging viral diagnosis with BSL-3 facilities were assigned as the first group of designated laboratories on January 22, 2020 after passing proficiency tests by Taiwan CDC. These eight designated laboratories and the three Taiwan CDC laboratories located in northern, central, and southern Taiwan formed the national diagnostic network and cumulatively delivered an initial diagnostic capacity of 500 tests per day (
During the COVID-19 global outbreak, Taiwan has further strengthened health security by implementing a strong and forward-looking diagnostic network to prevent and control outbreaks. Expanding the capacity of the laboratory network was required to meet the various diagnostic demands, such as extending travel history to other countries and enhancing screening for those who had severe complicated pneumonia, close contact with confirmed cases, passengers of airports or cruises, passengers from the Diamond Princess cruise ship, and three Wuhan special charter flights (
A second group of designated laboratories included three more BSL-3 laboratories and an additional 34 BSL-2 laboratories with negative air pressure facilities—were recruited for the response. A comprehensive diagnostic network with 60 laboratories was completed nationwide with a diagnostic capacity of 7,342 tests per day. In Taiwan, between January 14 and August 5 in 2020, 82,660 cases were reported, and 476 COVID-19 cases were confirmed by designated laboratories, including 7 deaths. Up to August 5, a total of 158,772 diagnostic tests were conducted (
COVID-19 is a category five notifiable disease in Taiwan which requires all cases fulfilling this requirement be reported to Taiwan CDC within 24 h. The turnaround time for laboratory testing results are provided in 24 h to isolate patients and trace close contacts in a cost-effective manner. COVID-19 control measures and patient management involve admitting confirmed patients in negative pressure isolation wards, home quarantine wards, and home isolation wards. These measures rely on laboratory testing—the timeliness of diagnosis results is therefore crucial for rational allocation of isolation wards and quarantine shelters and efficient use of medical resources, which can alleviate the burden on healthcare services. Proactive and targeted laboratory diagnosis of SARS-CoV-2 in Taiwan has proven to be efficient for real-time case recognition, contact tracing, and isolation. Due to the high infectivity of COVID-19 and the large proportion of patients with minor symptoms, laboratory testing with a 24-h turnaround time has been highly efficient in initiating a rapid control response. As of July 19, 2021, a network comprising a total of 226 designated laboratories conducting SARS-CoV-2 diagnosis was successfully established.
The Taiwan National Laboratory Network, which is experienced in dengue fever, avian influenza, MERS-CoV, and Zika diagnostic responses, faced a new challenge to meet the surge in demand for COVID-19 diagnostics. Laboratory surveillance needed to be further strengthened to prevent the risk of community transmission. To prepare for possible large-scale community outbreaks in the near future, further laboratory capacity will include more BSL-2 laboratories. In addition, more automatic high-throughput real-time PCR platforms as well as rapid nucleic acid and serological diagnostic tools will be integrated to expand screening capabilities. The advanced deployment of laboratory capacity for molecular diagnosis of SARS-CoV-2 made early recognition of COVID-19 cases possible and contributed to the containment and delay of community transmission in Taiwan. This strengthened laboratory capacity will help prepare for future challenges related to other emerging infectious diseases.
Our case histories of four HCBLs demonstrate their application, intent, design and utility to support laboratory diagnostic and reference activities and respond to surge demands such as COVID-19. Operating costs for HCBLs are high and industry experts estimated annual budgets for maintenance costs are 10–15% of the HCBL construction cost (
The successes of HCBLs and their trained personnel during the COVID-19 pandemic include several advancements of research and development, including supporting test and evaluation for laboratory diagnostics, vaccine studies, and related applications. The Kazakhstan Central Reference Laboratory, which is one of the newest BSL-3 laboratories, has performed impactful studies to support domestic vaccine development. Several international collaborations highlight their work that helps advance their products. MRIGlobal has supported test and evaluation for various government and commercial partners to aid in obtaining regulatory clearances for devices and assays. All of our case history laboratories also demonstrated laboratory diagnostic capabilities and ability to scale up operations during surge demands for COVID-19 testing.
In addition to increasing their capabilities and capacities, these HCBLs also adapted during the pandemic to meet changing demands during crisis moments. MRIGlobal and the Taiwan CDC cited BSL enhancements as one way to adapt existing laboratory infrastructure and increase capacity. Experts performed risk-based assessments prior to working in the BSL-2+ laboratories where they implemented modifications as well as additional training on PPE requirements and containment policies. CIRMF also had prior experience performing work in BSL-3+ and their BSL-4 was not fully used to support COVID-19.
The Taiwan CDC example highlights past preparations including avian influenza and MERS-CoV and a strong tiered network of laboratories. The capacity and capabilities within their BSL-3 laboratories provided guidance for laboratory networks at lower containment levels. The initial assessment of pathogens in BSL-3 also enabled the establishment of procedures to handle such pathogens at a lower containment level. These outputs serve as a model for pandemic response and mitigation.
While these case histories are a small representation of HCBL work, greater efforts are needed that continue to bring awareness and encourage transparency. The number of HCBLs continues to increase, that trend will likely continue with COVID-19 worldwide as countries and states will choose to prioritize and build them. Since many academic and private laboratories are not under their governmental oversight, it is difficult to obtain accurate counts of HCBLs (
While the value of HCBLs is established, the uncertain number of HCBLs also makes it difficult to ascertain capabilities and capacities for future preparedness. Regarding new and emerging pathogens, there is a need for higher biological containment for samples obtained from space exploration. These samples require containment to preserve their integrity while avoiding native Earth contamination of the sample and also to protect the Earth from extraterrestrial non-restricted or restricted bodies depending on where the sample is collected (
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.
KY and BS developed the concept and tables; KairT, FP, IM, KP, KaisT, AT, JRY, LG, and GO contributed content and developed revisions; SB reviewed, and all authors agreed to final version.
Author FP was employed by the company EpiPointe, LLC and author BS was employed by the company Joint Research and Development, Inc.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Special thanks to Denise M. Toney for her timely insights and thought leadership. The authors dedicate this work to Kay Mereish, a close and dear colleague, who would have contributed and encouraged this unfunded effort for its scientific impact and exemplar of building partnerships.